JPP60(1).book Page 15 Tuesday, November 27, 2007 1:25 PM

Research Papers

JPP 2008, 60: 15–20© 2008 The Authors
Received June 3, 2007
Accepted September 21, 2007
DOI 10.1211/jpp.60.1.0002
ISSN 0022-3573

Dosage plan of a flurbiprofen injection product using
inhibition of protein binding by lipid emulsion in rats
Kenji Ogata, Norito Takamura, Jin Tokunaga, Keiichi Kawai,
Kazuhiko Arimori and Shun Higuchi

Abstract

Second Department of ClinicalPharmacy, School of
Pharmaceutical Sciences, Kyushu
University of Health andWelfare, 1714-1 Yoshino,
Nobeoka, Miyazaki, 882-8508,
Japan
Kenji Ogata, Norito Takamura,Jin Tokunaga
Clinical Pharmacokinetics,
Division of Clinical Pharmacy,
Department of MedicoPharmaceutical Sciences, Facultyof Pharmaceutical Sciences,
Kyushu University, 3-1-1
Maidashi, Higashi-Ku, Fukuoka,
812-8582, Japan

Flurbiprofen-axetil (FP-ax), a bolus injection product of a non-steroidal anti-inflammatory drug
(NSAID), is a prodrug of flurbiprofen, an NSAID. As flurbiprofen strongly binds to site II of humanserum albumin (HSA), the free (unbound) concentration of flurbiprofen after injection of FP-ax islow. We have examined the inhibitory effect of free fatty acid (FFA), a binding inhibitor for site II of
HSA, on the binding of flurbiprofen in-vitro and in-vivo by ultrafiltration, to establish an effective
dosage of FP-ax. In-vitro, fatty acid mixtures (FAs) inhibited the binding of flurbiprofen to rat serum
albumin. The free fraction of flurbiprofen was remarkably increased by FAs in rat serum. In-vivo, FPax was injected into a control group (low FFA concentration in serum) and a lipid emulsion group(high FFA concentration in serum). The area under the curve of the free concentration of flurbiprofen during the alpha phase and the distribution volume of the central compartment of flurbiprofen
were significantly higher in the lipid emulsion group than the control group (5.0- and 1.2-times,
respectively). When FP-ax was administered at high FFA concentration, the free concentration of
flurbiprofen and distribution of flurbiprofen to tissues increased transiently. This administration
method may be useful for patients with cancer pain, having a potent analgesic effect.

Introduction

Kenji Ogata, Shun Higuchi
School of Health Sciences,
Faculty of Medical, Kanazawa
University, 5-11-80 Kadatsuno,
Kanazawa, Ishikawa, 920-0942,
Japan
Keiichi Kawai
Department of Pharmacy,
Miyazaki Medical College
Hospital, 5200 Kihara, Kiyotake,Miyazaki, 889-1692, Japan
Kazuhiko Arimori

Correspondence: N. Takamura,
Second Department of ClinicalPharmacy, School of
Pharmaceutical Sciences, Kyushu
University of Health andWelfare, 1714-1 Yoshino,
Nobeoka, Miyazaki 882-8508,
Japan. E-mail:noritotaka@phoenix.ac.jp
Funding: This research was
supported in part by aGrant-in-Aid from the Programfor Strategic Regional Scienceand Technology Advancement,
MiyazakiCurrently, the guidelines of the World Health Organization for relieving cancer pain with
analgesics advocate a so-called ‘analgesic ladder’: patients with mild pain (step 1) should betreated with non-steroidal anti-inflammatory drugs (NSAIDs), patients with moderate pain
(step 2) and severe pain (step 3) should receive an opioid in combination with NSAIDs(World Health Organization 1986). Thus, NSAIDs are important for reducing cancer pain
(Pace 1995; Cherny 2000).
The intravenous administration of NSAIDs is expected to have a rapid and potent analgesic effect. Flurbiprofen-axetil (FP-ax), a bolus injection product of NSAID, is a prodrugof flurbiprofen. FP-ax is rapidly hydrolysed in flurbiprofen by esterase in blood. As theanalgesic effect of FP-ax is rapid and continuous, the drug is indispensable for relieving
cancer pain. Flurbiprofen binds to human serum albumin (HSA) very strongly, its association constant is approximately 1.0 ×10−7 M−1 (Takla et al 1985), and so the free (unbound)concentration of flurbiprofen in serum is low. In general, since the pharmacological effectof a drug depends on its free concentration (Dasgupta 2002), it seems that the analgesic
effect of flurbiprofen is not sufficiently exerted. We reported that bucolome, an NSAID,
inhibited furosemide, which is a diuretic binding to site I of HSA, increasing the free concentration of furosemide in serum and urine, and as a result, restoring the diuretic response
to furosemide (Takamura et al 2005). Flurbiprofen binds to site II of HSA (Sudlow et al
1976; Wanwimolruk et al 1983). If some drugs can temporarily inhibit the binding of flurbiprofen to site II using the previous inhibition method (furosemide–bucolome–HSA interactions), the free concentration of flurbiprofen in serum will be increased and the analgesic
effect may be enhanced.
A lipid emulsion is often administered to patients with cancer (Dionigi et al 1985). Free
fatty acid (FFA) is released from the emulsion by lipoprotein lipases in blood. The FFA
mainly consists of long chain fatty acids such as oleic acid and linoleic acid. FFA binds tosite II of HSA (Menke et al 1989); however, a study of flurbiprofen–FFA–HSA interactions

15

JPP60(1).book Page 16 Tuesday, November 27, 2007 1:25 PM

16

Kenji Ogata et al

has not been reported. To establish an effective dosage of FPax, we have examined the inhibitory effect of FFA on thebinding of flurbiprofen in rats.

The free fraction of flurbiprofen was calculated by dividing the unbound flurbiprofen concentration by the total(bound + unbound) flurbiprofen concentration:

Materials and Methods

free fraction (fu) =Materials

Flurbiprofen powder and flurbiprofen-axetil (10 mgmL−1)
were donated by Kaken Pharmaceutical Co. (Tokyo, Japan).
Oleic acid and linoleic acid were purchased from Nacalai
Tesque, Inc. (Kyoto, Japan). Intralipos (refined soybean oil
20%, concentrated glycerin 2.2% and refined egg yolk
lecithin 1.2%), a lipid emulsion, was obtained from Otsuka
Pharmaceutical Co. (Tokyo, Japan). HSA (essentially fatty
acid free) was purchased from Sigma-Aldrich (St Louis, MO,
USA). All other reagents were of analytical grade. A 0.067 M
phosphate buffer (pH 7.4) was prepared with dibasic sodium
phosphate and monobasic sodium phosphate.
Rat serum was pooled from three adult male Wistar rats
(380–400 g). Rats were anaesthetized with ether. Blood samples were drawn from the abdominal aorta with polyethylene
disposable syringes and transferred into vacutainers. The
blood samples were immediately centrifuged and the serum
was separated. The concentration of rat serum albumin (RSA)in this serum was 525 mM. HSA solution (600 mM) was prepared with 0.067 M phosphate buffer (pH 7.4). Fatty acid mixtures (FAs) were prepared by mixing oleic acid and linoleic
acid ([linoleic acid]/[oleic acid] = 2).
Binding experiments in-vitro

In-vitro binding of flurbiprofen to albumin (RSA or HSA)
was determined in rat serum and in isolated HSA solution at
25°C by the following procedures. The concentration of flurbiprofen was 10 mgmL−1 (40 mM), corresponding to the maximum therapeutic concentration. To assess the inhibitory
effect of FAs on the binding of flurbiprofen to RSA, FAs
were added to rat serum to give final concentrations of 2625–
7875 mM – multiples of the molar concentration of RSA. To
study the inhibitory effect of oleic acid on the binding of flurbiprofen to HSA, oleic acid was added to isolated HSA solution to give final concentrations of 300–2400 mM – multiplesof the molar concentration of HSA. Free species of flurbiprofen were prepared by ultrafiltration (1670 g, 8 min, 25 °C)
using a Tosoh plastic ultrafiltration apparatus, Ultracent-10(Tosoh Co., Tokyo, Japan). Adsorption of flurbiprofen onto
the membrane or apparatus was negligible.
Free concentrations of flurbiprofen were determined by
HPLC using a system consisting of a RF-10AXL fluorescence
detector, a LC-10ADvp pump, a SIL-10ADvp auto injector, aSCL-10Avp system controller, and a CTO-10Avp column
oven, all from Shimadzu (Kyoto, Japan). A LiChrospher RPSelect B column (5 mm) (Kanto Reagents, Tokyo, Japan) wasused for the stationary phase. Flurbiprofen was assayed byfluorescence monitoring (ex. 248 nm, em. 320 nm). HPLC
was performed at a flow rate of 1.0 mL min−1 at 40°C, using
0.1 M citric acid–0.1 M sodium acetate (pH 4.5)/acetonitrile(1:1, v/v) as the mobile phase.

[ Df ][ Df ] + [ Db]

where [Df] and [Db] are unbound and bound drug concentrations, respectively.
Pharmacokinetics of flurbiprofen in rats

The protocol of the animal study was approved by the Animal
Care and Use Committee of the Kyushu University of Healthand Welfare. Adult male Wistar rats (327–408 g) were housedin an air-conditioned room with free access to commercial
feed and water. Rats were anaesthetized with a 2 mLkg−1 urethane solution (60%, w/v). A cannula was inserted into thefemoral vein and artery using polyethylene tubing (i.d.
0.5 mm; o.d. 0.8 mm; Natsume Seisakusyo, Tokyo, Japan).
The body temperature of the rats was maintained by heatingwith a lamp. To adjust the solution for intravenous injection
to a dose of 1.0 mgkg−1, the FP-ax solution was diluted withsaline. FP-ax was injected into two groups of rats intravenously. Saline (3 mLkg−1) was administered to one group(control group), 30 min before the injection of FP-ax. A lipid
emulsion (Intralipos, 3 mLkg−1) was administered to theother group (lipid emulsion group) to increase the serum concentration of FFA, 30 min before the injection of FP-ax. After
FP-ax injection, the cannula was flushed with a small volumeof heparinized saline to ensure that the complete dose had
been administered and to prevent the formation of clots.
Blood samples (600 mL) for the measurement of flurbiprofen and FFA concentrations were taken from the femoral
artery at 1, 5, 10, 30, 60 and 180 min after the administrationof FP-ax. To obtain serum, the blood sample was placed in
Capiject (Terumo Medical Co., USA) and centrifuged(1200 g for 10 min).
Serum concentrations of flurbiprofen (bound + unbound)
were estimated by the following procedure. Serum (30 mL) was
added to 50 mL0.01 M NaOH (containing 3 mgmL−1 of 4-biphenylacetic acid, i.s.), 100 mL 3 M HCl, and 1.5 mL cyclohexane,
shaken for 10 min, and centrifuged. A 20-mL organic layer
sample was injected into the HPLC system. Free concentrations of flurbiprofen were estimated as follows. Serum(250 mL) was placed in an ultrafiltration apparatus, Minicent-10(Tosoh Co., Tokyo, Japan). The filtrated solution (20 mL) was
added to 50 mL 0.01 M NaOH (containing 3 mg mL−1 4-biphenylacetic acid, i.s.), 100 mL 3 M HCl, and 1.5 mL cyclohexane,
shaken for 10 min, and centrifuged. A 1.0-mL organic layer
sample was evaporated and resolved by HPLC mobile phase(70 mL) and the sample was injected into the HPLC system(50 mL). HPLC conditions were as described above in-vitro.
Pharmacokinetic analysis

Total (bound + unbound) and free areas under the concentrationof the flurbiprofen–time curve from 1 to 10 min (AUC1-10,totaland AUC1-10,free) after the administration of FP-ax wereJPP60(1).book Page 17 Tuesday, November 27, 2007 1:25 PM

17

Coadministration of flurbiprofen and lipid emulsion product

Vdc = DoseFP/C1 min
where DoseFP is the dosage of flurbiprofen calculated basedon molecular weight (flurbiprofen, 244.26; FP-ax, 330.36)
from the dosage of FP-ax. C1 min was the maximum concentration of flurbiprofen hydrolysed from FP-ax in serum.
Measurement of albumin and FFA concentrations

Albumin and FFA were measured by the bromcresol green
method (Doumas et al 1971) and enzyme method (Shimizu
et al 1979), respectively, using Cobas Integra 400 plus (Roche
Diagnostics, Basel, Switzerland). The assay kits for albumin
and FFA were ALB II (Roche Diagnostics, Basel, Switzerland) and Nescauto NEFA-V2 (Alfresa Pharma Co., Osaka,
Japan), respectively.

a,b,c

0.15

Free fraction of flurbiprofen

calculated using the log trapezoidal rule. The distribution volume of the central compartment (Vdc) of flurbiprofen was
calculated with the following equation:

a,b,d
0.1

0.05

a,c,d

0

A

B

C

D

Figure 1 Effect of oleic acid and linoleic acid mixtures (FAs) on thefree fraction of flurbiprofen in rat serum: A, control; B, [FAs]/[RSA] = 5; C, [FAs]/[RSA] = 10; D, [FAs]/[RSA] = 15. The following
concentrations were used: rat serum (as RSA), 525 mM; flurbiprofen,
10 mg mL−1. Each column is the mean of three experiments ± s.d.
a
P < 0.001 vs A; bP < 0.001 vs B; cP < 0.001 vs C; dP < 0.001 vs D.

Student’s t-test was used to analyse differences between two
groups. Analysis of variance was used to analyse differencesamong more than two groups, and the significance of difference between two means in these groups was evaluated using
Fisher’s protected least significant difference.

Results
Effect of oleic acid and linoleic acid on thebinding of flurbiprofen in rat serum

To examine FFA as a potential inhibitor of the binding of flurbiprofen to RSA, we measured the free concentration of flurbiprofen
and calculated the free fraction of flurbiprofen. In-vivo, we
examined the inhibitory effect of FFA using a lipid emulsion,
but in-vitro, the inhibitory effect of FFA released from a lipid
emulsion could not be evaluated because the activity of lipoprotein lipase was weak in the rat serum added to the lipid emulsion. Therefore, FAs that mimic soybean oil, which is the main
component of lipid emulsion, were used in-vitro. To adjust to5-, 10- and 15-times the RSA concentration ([FAs]/[RSA] were5, 10 and 15), FAs were added to rat serum containing flurbiprofen (10 mgmL−1). Figure 1 shows that the free fraction of
flurbiprofen increased significantly with increasing FAs. When[FAs]/[RSA] was 5, the free fraction of flurbiprofen wasapproximately 20.9-times that of the control. Further addition of
FAs to serum increased the free fraction of flurbiprofen remarkably, the value being approximately 83.7-times that of the control at [FAs]/[RSA] =15. This in-vitro study indicated that thebinding of flurbiprofen to RSA was inhibited by FAs.
Effect of FFA on the pharmacokinetics offlurbiprofen in rats

To evaluate the effect of FFA on the pharmacokinetics offlurbiprofen, FP-ax was injected in the control group andSerum concn of free fatty acid (μ M)

Statistical analysis

10000

∗∗∗
∗∗∗
8000
∗∗∗

∗∗∗

6000
4000
2000
0

0

50

100
Time (min)

150

200

Figure 2 Serum concentration of free fatty acid–time profiles after
FP-ax injection to the control group (䊊) and lipid emulsion group (䊉).
Each point represents the mean ± s.d. (n = 6). ***P < 0.001 comparedwith the control group.

lipid emulsion group. Figure 2 shows the time course of thechange in serum concentration of FFA after the injection of
FP-ax in the two groups. Serum concentrations of FFA weresignificantly higher in the lipid emulsion group than the control group until 30 min after the administration of FP-ax, andthere were no significant differences in the FFA concentrations of the two groups at 60 min after the injection.
Figure 3 indicates the time course of the change in serum
concentration of flurbiprofen after the injection of FP-ax inthe two groups. During the alpha phase (until 10 min after FPax administration), serum concentrations of flurbiprofen werelower in the lipid emulsion group than the control group.
Figure 4 shows the time course of the change in free concentration of flurbiprofen after the injection of FP-ax in thetwo groups. As in the serum FFA profile, free concentrationsof flurbiprofen were significantly higher in the lipid emulsion

JPP60(1).book Page 18 Tuesday, November 27, 2007 1:25 PM

18

Serum concn of flurbiprofen (μ g mL–1)

Kenji Ogata et al(AUC1-10,total) in the lipid emulsion group was significantly
lower than the value obtained from the control group. The
AUC of free concentrations of flurbiprofen during the alphaphase (AUC1-10,free) was remarkably higher in the lipid emulsion group than the control group. The ratio of AUC1-10,free inthe lipid emulsion group to AUC1-10,free in the control group
was approximately 5-times. The distribution volume of thecentral compartment (Vdc) of flurbiprofen was significantly
larger in the lipid emulsion group than the control group.

100

∗∗∗

10

1

Discussion
0

50

100
Time (min)

150

200

Free concn of flurbiprofen (μ g mL–1)

Figure 3 Serum concentration of flurbiprofen–time profiles after FPax injection to the control group (䊊) and lipid emulsion group (䊉). Eachpoint represents the mean ± s.d. (n = 6). ***P < 0.001 compared with thecontrol group.

10

∗∗∗
∗∗∗
∗∗∗ ∗∗∗

1
0.1
0.01
0.001

0

50

100

150

200

Time (min)Figure 4 Free concentration of flurbiprofen–time profiles after FP-ax
injection to the control group (䊊) and lipid emulsion group (䊉). Eachpoint represents the mean ± s.d. (n = 6). ***P < 0.001 compared withthe control group.

group than the control group until 30 min after the administrationof FP-ax, and there were no significant differences in freeflurbiprofen concentrations of the two groups at 60 min after
the injection.
Table 1 shows the pharmacokinetic parameters of flurbiprofen obtained from the alpha phase. The AUC of serum
concentrations of flurbiprofen during the alpha phase

Table 1 Kinetic parameters obtained from flurbiprofen concentrations
during the alpha phase after FP-ax injection in the control group andlipid emulsion group
Parameter−1

Vdc (L kg )AUC1–10, total (mg min mL−1)
AUC1–10, free (mg min mL−1)

Control

Lipid emulsion

0.077 ± 0.009
66.4 ± 5.34
1.35 ± 0.94

0.092 ± 0.007**
60.0 ± 3.09*
6.71± 1.00***

Each value represents the mean ± s.d. (n = 6). *P < 0.05, **P < 0.01,***P < 0.001 compared with the control group.

To administer FP-ax with an effective dosage plan, a way toincrease the free concentration of flurbiprofen in serum isrequired. In this study, the inhibitory effect of FFA on thebinding of flurbiprofen to RSA was investigated. When FFA
inhibited flurbiprofen binding to RSA in-vivo, the serum concentrations of flurbiprofen during the alpha phase changed
markedly; therefore, we mainly observed the pharmacokineticsof flurbiprofen during the alpha phase. The free concentrations and Vdc values obtained from the pharmacokinetics offlurbiprofen during the alpha phase were higher in the lipid
emulsion group than the control group (Figure 4 and Table 1).
This indicated that the inhibition of flurbiprofen bound to
RSA by FFA could allow flurbiprofen to distribute to the target tissues. It may be speculated that the concentration of
FFA was increased by injecting a lipid emulsion in rats at thetime when FP-ax was administered, FFA inhibited the binding of flurbiprofen to RSA, and as a result, flurbiprofen was
distributed to the target tissues.
In general, if the free concentration of a drug is increased
remarkably by inhibiting protein binding, the serum concentration is decreased (Arimori et al 1984). This study showedthat serum concentrations of flurbiprofen during the alphaphase were significantly lower in the lipid emulsion groupthan the control group, whereas those during the beta phasewere slightly higher without a significant difference in thelipid emulsion group (Figure 3). This result would be dueto the higher concentration of RSA in the lipid emulsion
group (lipid emulsion group, 445 ± 25 mM; control
group, 371 ± 13 mM). It could be speculated that the retentionof flurbiprofen in serum would be increased during the betaphase, in which the affinity of flurbiprofen was increased by
reducing the concentration of FFA, a site II binding inhibitor;
however, it is notable that the binding of flurbiprofen to RSA
was strongly inhibited by FFA during the alpha phase, though
the RSA concentration was higher in the lipid emulsion groupthan the control group.
We revealed that the free fraction of flurbiprofen increased
significantly as the binding of flurbiprofen to site II of HSA
was inhibited by oleic acid, which is abundant in human serum(control, free fraction = 4.9 × 10−5 ± 0.1 × 10−5; [oleic acid]/[HSA] = 2, free fraction = 6.6 × 10−5 ± 0.2 × 10−5, P < 0.05 vscontrol; [oleic acid]/[HSA]=3, free fraction=13.1×10−5± 0.1 × 10−5, P < 0.01 vs control; [oleic acid]/[HSA] = 4, free
fraction=23.9 × 10−5 ± 0.7 × 10−5, P < 0.01 vs control, eachvalue is the mean of three experiments±s.d.). It was reported
that HSA has six to seven different binding sites of FFA andthe second binding site of FFA corresponds to site II (Petitpas

JPP60(1).book Page 19 Tuesday, November 27, 2007 1:25 PM

Coadministration of flurbiprofen and lipid emulsion productet al 2001; Chuang & Otagiri 2002). Arg410 and Tyr411,
which are amino acid residues in site II, interact electrostatically with the carboxy group of FFA (Bhattacharya et al
2000; Petitpas et al 2001). These amino acid residues play animportant role in the binding of ketoprofen, which is an
NSAID of the arylpropionic acid class involving flurbiprofen
and has a carboxy group (Watanabe et al 2000). Here, theidentity of the amino acid sequence, which is the primarystructure, between HSA and RSA was high, and Arg410 and
Tyr411 in site II of HSA exist in the RSA binding site (Carter& Ho 1994); therefore, the binding inhibitory mechanismof flurbiprofen by FFA was speculated to be similar in HSA
and RSA.
When a lipid emulsion is administered to patients, theserum concentration of FFA can increase to approximately
3000 mM, comparable with 5-times the HSA concentration(Zimmerman et al 1981). The increment of FFA concentration
can inhibit flurbiprofen binding to HSA. It was predicted thatthe injection of FP-ax would lead to an increase in the free
concentration of flurbiprofen when FFA concentration wasincreasing by administering a lipid emulsion. Though a lipid
emulsion was used in this study to increase the serum concentration of FFA, there are other ways to increase FFA concentrations. For example, the injection of heparin, an
anticoagulant drug, to patients at risk for thrombus formation
activates lipoprotein lipase, and can increase the FFA concentration transiently by approximately 3000 mM (Naranjo et al
1980; Brown et al 1981). Furthermore, the FFA concentration
fluctuates in a normal physiological state and is generally highduring fasting (Reaven et al 1988; Wolever et al 1995). FP-ax
can be administered with the result of increasing FFA concentrations by heparin injection for ‘anticoagulant’ therapy andfasting. So far, we have elucidated that when a diclofenac suppository (NSAID: distribution volume is small; binding affinity of HSA is high) was administered to patients withrheumatoid arthritis at high FFA concentration during fasting,
FFA inhibited the binding of diclofenac to HSA, and the analgesic effect of diclofenac increased (Takamura et al 2007).
The control of cancer pain has tended to involve opioids
alone, and many patients are supported with high doses of
opioids; however, administering a combination of NSAID
and opioids has an opioid-sparing effect as well as better
analgesia (Bjorkman et al 1993; Joishy & Walsh 1998; Zelcer
et al 2005). Combination therapy prevents side effects such as
severe constipation and somnolence which are induced byexcess opioids; therefore, it is important to establish aneffective dosage plan for FP-ax to enhance the analgesic
effect of NSAIDs.
We elucidated that when FP-ax was administered at a high
FFA concentration, the free concentration of flurbiprofen anddistribution of flurbiprofen to tissues increased transiently.
This administration method may give patients with cancer
pain a potent analgesic effect.

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